The present invention relates to a scanning cytometer for acquiring cytometric data of a cell population as a sample by scanning and measuring the cell population with optical beams in accordance with measuring conditions such as a measuring range and measuring speed for the sample.
There have been flow cytometers which are apparatuses for irradiating individual cells in a cell population of a living organism biochemically identified by a fluorescent pigment with a laser to form laser spots where the cells are excited to emit fluorescent or scattered light, measuring the light at a high speed and analyzing the results of the measurement to acquire and present statistic data as cytometric data representing the immunological, genetic and proliferative characteristics of the cell population.
The laser of such a flow cytometer and spots formed by converging laser beams therefrom are fixed. Cells are measured on such fixed spots using converged beams by causing the cells to flow through the spots along with a jetted stream of water in a state in which the individual cells are floating apart from each other in a drifting liquid. With such a method of measurement, it is not possible to find the cell associated with a particular item of data to observe the state of the cell and to identify measurement the data associated with each cell after the measurement.
An alternative to such flow cytometers, scanning cytometers have been developed including the invention disclosed in Jpn. Pat. Appln. KOKAI Publication No. 3-255365 titled "Method and Apparatus for Measuring a Plurality of Optical Characteristics of Living Sample" wherein spots formed by converged laser beams are scanned across a cell population on a slide glass to detect fluorescent and scattered light emitted by individual cells in the cell population and wherein resultant data are processed.
In such a scanning cytometer, scanning is performed with a scanner driven in accordance with a scanning waveform, etc. which deflects laser beams, e.g., a focused spot at X-direction and moves a moving scanning stage at Y-direction to scan a cell population on the slide glass on a moving scanning stage. Fluorescent and scattered light emitted by cells excited by the laser beams as spots is converted into electrical signals which are collected in accordance the logic of a data input signal output along with the scanner scanning waveform.
A scanning cytometer processes images formed as a result of scanning, extracts measurement data on each cell such as the sum of the values of fluorescent light, area of the cell, the maximum value of fluorescent light, coordinates on the scanning stage, elapse time since the beginning of the measurement, the distance of the cell to the nearest cell, the circumference of the cell and the number of spots in the cell and processes such data statistically to provide the results to the measuring personnel (operator) through an interface such as a computer.
Since a tremendous number of cells are processed resulting in a very wide measuring area, it is not possible to collect such data at a time. Therefore, the measuring area is divided into small ranges referred to as "strips", and measurement is performed on each of such strips.
Scanning cytometers are significantly different from flow cytometers in that they have a function referred to as "recall function" which makes it possible to retrieve each cell of interest from statistic data obtained as described above. When measurement is carried out using a scanning cytometer, it is necessary to set measuring conditions prior to the measurement. The measuring conditions e.g., a voltage applied by a photomultiplier (PMT) for converting fluorescent light emitted by cells into electrical signals and an offset adjusting voltage of the photomultiplier, the gain and the offset for a photodiode (PD) as a detector for converting scattered light into electrical signals, the measuring area, contouring threshold and minimum cell area.
Those measuring conditions are basically set by the operator at values which are desired or determined as appropriate by the operator.
A description will now be made with reference to FIGS. 1A and 1B on how to set, for example, the application voltage of the PMT of a conventional scanning cytometer and the offset adjusting voltage of the same. There are two methods to set the application voltage and offset adjusting voltage of the PMT.
As shown in FIG. 1A, the first method is to obtain a fluorescent image for one strip by performing measurement with the application voltage and offset adjusting voltage of the PMT set at appropriate initial values. The resultant fluorescent image of a cell is examined to obtain the brightness of the fluorescent light emitted by the cell and the brightness of the background and, if the brightness is not a proper value or is out of a proper range, the application voltage and offset adjusting voltage of the PMT are reset according to the judgment of the operator. An optimum value is determined after repeating such measurement, judgment and resetting several times.
As shown in FIG. 1B, the second method is to scan a single line and to perform a photometric process with the application voltage and offset adjusting voltage of the PMT set at appropriate initial values by only deflecting laser beams projected upon an arbitrary cell population without moving the scanning stage. The result of the photometric process can be presented to the operator real time. The operator adjusts the application voltage and offset adjusting voltage of the PMT by trial and error based on the result of photometry such that the brightness of fluorescent light emitted by the cells and the brightness of the background assume appropriate values or stay within appropriate ranges.
A description will now be made with reference to FIGS. 2A and 2B on methods for setting the measuring area of a conventional scanning cytometer. There are two methods for setting the measuring area.
As shown in FIG. 2A, according to the first method, the operator directly inputs the values of the coordinates of the starting and end points of measurement on a computer.
As shown in FIG. 2B, according to the second method, the operator moves the scanning stage while observing the sample, stops the scanning stage at an appropriate position and specifies the location (point) to start measurement. Then, the operator moves the scanning stage again while observing the sample, stops the scanning stage at an appropriate position and specifies the location (point) to end the measurement.
Two problems arise as described below when the application voltage and offset adjusting voltage of the PMT are set in a conventional scanning cytometer.
The first problem is wasteful time and labor spent by the operator during condition setting repeated several times by trial and error.
The second problem arises in that there is no assurance that conditions for measurement are appropriate in obtaining data for cells in a wide area in actual measurement because the measurement conditions are set based on brightness data of cells or a cell population in a range as small as one strip or one line. Specifically, let us assume that the cell cycle of a cell in the process of cell division is actually measured on a single line scan with the application voltage and offset adjusting voltage of the PMT set at appropriate values for a cell in a stable state. A cell in the process of cell division emits a greater amount of fluorescent light than a cell in a stable state because of a difference in the amount of DNA. As a result, when the cell in the process of cell division is measured with the application voltage and offset adjusting voltage of the PMT set at appropriate values for the cell in a stable state, the brightness of fluorescent light from the cell in the process of cell division can exceed the measuring area. Measurement must be redone when the measuring area is exceeded.
Although the first problem can be solved by an existing process of automatically adjusting the application voltage and offset adjusting voltage of a PMT employed in industrial scanning microscopes or scanning microscopes for living organisms, the second problem remains unsolved.
Even if the existing process for automatically adjusting the application voltage and offset adjusting voltage of a PMT is applied to an actual measuring area instead of a small area such as one strip or one line to solve the second problem, another problem arises in that a long time is spent for setting conditions because the area is widened.
A problem occurs as described below when a measuring area is set for a conventional scanning cytometer depending on how the operator sets the measuring area on the sample. A sample observed on a scanning cytometer is a population of cells colored with a fluorescent pigment such as a smear of floating cell sap or a touch smear of organic cells placed on a slide glass which is manually prepared. The cells can be unevenly distributed on the slide glass or variation can occur in fluorescent coloring depending on the manner in which the sample is prepared.
During the measurement of such a sample, the efficiency of cell measurement and the accuracy of measurement and analysis can be reduced when the measuring area is set based on subjective judgment of the operator as in the prior art.
The reason is that the operator can not know the state of distribution of a cell population across a wide area on the slide glass and the degree of the uniformity of fluorescent coloring in a conventional scanning cytometer. It can happen that a region including a small number of cells, a region where an agglomeration of cells is formed, or a region having variation of fluorescent coloring is set as the measuring area. The efficiency of measurement is reduced if a region including a small number of cells is measured. The measuring efficiency is also reduced in a region where cells concentrate so densely that a cell agglomeration is formed. When a region having variation of fluorescent coloring is measured, the. distribution of the sum of values of fluorescent light from individual cells can be dependent not only on the amount of cellular components such as DNA but also on variation of fluorescent coloring, and the result of measurement and analysis using a cytometer includes artifacts that are attributable to the dependence on the variation of fluorescent coloring.
Attempts to obtain quantitative knowledge of the state of distribution of a cell population across a wide area and the degree of uniformity of fluorescent coloring will not be practical because such measurement over a wide area will take an enormous amount of time if it will be carried out similarly to normal measurement.
It is an object of the present invention to provide a scanning cytometer in which conditions for measurement such as a gain and an offset of a detector and a measuring area can be automatically set based on cell data in a wide area.